Fluorescent labeling reagents with multiple donors and acceptors

ABSTRACT

Disclosed is a novel class of fluorescent resonance energy transfer (FRET) labelling reagents, based on and synthesised from easily prepared dye building blocks. The labelling reagents are in the form of “cassettes” which enable their attachment to a wide variety of biological and other materials. A labelling reagent comprises at least two fluorescent dye moieties covalently linked via a linker group and optionally having a target bonding group for attaching the reagent to a target. The energy transfer labelling reagents may be bound to target materials through covalent or non-covalent attachment. The dyes are selected so that the emission spectrum of a first (or donor) dye overlaps the absorption spectrum of a second dye, thereby allowing energy transfer to occur between the dyes. The dye building blocks are 4′, 5′-bis-aminomethyl-fluorescein and/or its 5(6)-carboxylic acid and having the structure (1). In addition to the embodiment of the invention which includes a single donor and a single acceptor fluorochrome, the fluorescent energy transfer labelling reagents according to the invention may further comprise one or more third fluorochromes each having third absorption and emission spectra covalently attached to said first or second fluorochromes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/413,517, filed Sep. 25, 2002; the disclosures of which areincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to fluorescent dyes, more particularly toenergy transfer fluorescent dyes with multiple donors and/or acceptorsand their applications.

DESCRIPTION OF RELATED ART

A variety of fluorescent dyes have been developed for labeling anddetection of components in biological and other systems. One class ofdyes developed and applied extensively to DNA sequencing is fluorescenceresonance energy transfer (FRET) based fluorescent dyes, which areconstructed of a donor dye and an acceptor dye. Generally, in thesedyes, the donor and the acceptor dyes are positioned in close proximityand with proper orientation to each other, the photon energy absorbed bythe donor is transferred to the acceptor causing the acceptorfluorophore to fluoresce when excited at the donor absorptionwavelength. To ensure the most efficient transfer of energy, it isimportant that the donor fluorophore has high extinction coefficient,high quantum yield and efficient transfer of the absorbed excitationenergy of the donor to the acceptor in the form of acceptor fluorophoreemission. Furthermore, for efficient energy transfer, there should begood overlap between the emission of the donor dye and the absorption ofthe acceptor dye.

A variety of energy transfer fluorescent dyes, mostly involving twofluorophores (as donors and acceptors), have been described in theliterature (Proc. Natl. Acad. Sci, USA (1995) 92, 4347-4351, Anal.Biochem. (1995), 231, 131-140, Nucleic Acids Research (1996), 24,1144-1148, Anal. Biochem. (1996), 243, 15-27, Nucleic Acids Research(1997), 25, 2816-2822, and Tetrahedron Letters (2000) 41, 8867-8871).However, as mentioned previously, the energy transfer is a function ofspectral overlap between the emission of the donor and the absorption ofthe acceptor. When such an overlap is marginal, as shown by fluoresceinand a much longer wavelength absorbing fluorescent dye such as Cy5™(with the emission of fluorescein at 520 nm and the absorption of Cy5 at650 nm), a low ET results. In such a case, two consecutive energytransfer processes with better spectral overlaps could be moreefficient.

This kind of strategy led to recent literature precedence (U.S. Pat. No.6,008,373 and U.S. Pat. No. 6,130,094) to two consecutive energytransfer process involving fluorescent dyes. Waggoner and co-workers(U.S. Pat. No. 6,008,373) reported an example of two consecutive energytransfers, one from fluorescein to a Cy3 followed by another transfer ofenergy from the Cy3 to an attached Cy5, which gave a large Stokes' shiftof 172 nm. They obtained an even larger Stokes' shift of 282 nm when Cy7was used as the longest emitting fluorophore. However, no conclusion wasmade whether two consecutive energy transfer steps were more efficientthan a direct one, for instance, from fluorescein to Cy5 or Cy7.

Ju et al (J. Am. Chem. Soc., (2001), 123, 12923-12924 and WO 02/22883)constructed a trichromophore-labeled oligonucleotide that had a scaffoldof 26 nucleotides, designated as F-4-R-6-Ct-13. When excited at 488 nm,the predominating emission of the assembly was at 670 nm due to Cy5 anda Stokes' shift of 182 nm with an overall quantum yield of 0.13, whilethe quantum yield of the Cy5 was 0.27. However, since the extinctioncoefficient of Cy5 at 488 nm was much less than at peak absorption of650 nm (less than 2%), this assembly was much brighter than theunmodified dye by a factor of at least 25 when excited at 488 nm.

Despite its usefulness in labeling primers for DNA sequencing andPCR-based genetic analysis, such an assembly cannot be used as alabeling reagent for much more general usage. In this invention, we havedesigned a labeling reagent with molecular architecture based on4′,5′-bis-aminomethyl fluorescein as shown in FIG. 1. This moleculardesign provides close to optimal spacing between donor and acceptorfluorophores and, hence, high energy transfer (ET) efficiency.

The basis of energy transfer is generally accepted as Forster ResonanceEnergy Transfer (FRET) by a dipole-dipole interaction mechanism proposedby Theodor Forster (Joseph R. Lakowicz, “Principles of FluorescenceSpectroscopy” 2^(nd) Edition, Chapter 13, Kluwer Academic PlenumPublishers, 1999). Forster's theory implies that the closer the donorand the acceptor fluorophores, the better the energy transfer. However,experience has shown that these two fluorophores should not be too closeto cause quenching of each other.

Therefore, as in most practical ET applications, linkers were used tokeep the fluorophores separated as illustrated in U.S. Pat. No.5,863,727 and WO 00/13026. This approach was adopted, despite the factthat the introduction of these linkers eventually lengthened the spatialseparation between the two fluorophores, and, thus, lowered theefficiency of energy transfer.

In reported literature (Nucleic Acids Research, (1997), 25, 2816-2822),the most optimal separation between donor and the acceptor fluorophoresappears to be that shown by the “bifluor-1”, a dye dimer consisting of5-carboxytetramethyl-rhodamine linked to 4′-aminomethylfluorescein-5-carboxylic acid. However, “bifluor-1” was not used in DNAsequencing due to considerations such as poor enzyme incorporation andothers.

Moreover, the “bifluor-1” structure cannot be used for the transfer ofenergy between three fluorophores since, after the attachment of twofluorophores onto 4′-aminomethyl fluorescein-5 carboxylic acid, there isno functional group left for the attachment of a biological molecule,such as a nucleotide.

For this reason, we have developed a 4′,5′-bis-aminomethyl-fluoresceinas the basic skeleton for our “trifluor-1”. Fluorescent labels based onsuch a “trifluor-1” structure can be excited optimally at a neon-argonlaser at a wavelength of 488 nm and fluoresce at a significantlydifferent wavelength with large Stokes' shifts. Also, with theintroduction of a 5- or 6-carboxyl group, the basic structure can beextended in its use for synthesising a fluorescent labeling reagent forvarious applications.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a novel class of fluorescentresonance energy transfer (FRET) labelling reagents, based on andsynthesised from easily prepared dye building blocks. The labellingreagents are in the form of “cassettes” which enable their attachment toa wide variety of biological and other materials. A labelling reagentcomprises at least two fluorescent dye moieties covalently linked via alinker group and optionally having a target bonding group for attachingthe reagent to a target. The target bonding group is chosen to besuitable for forming a covalent linkage with a complementary group onthe target material. Alternatively, the energy transfer labellingreagents may be bound to target materials through non-covalentattachment. The dyes are selected so that the emission spectrum of afirst (or donor) dye overlaps the absorption spectrum of a second dye,thereby allowing energy transfer to occur between the dyes. The dyebuilding blocks are 4′,5′-bis-aminomethyl-fluorescein and/or its5(6)-carboxylic acid and having the structure (I).

Thus, in a first aspect there is provided a compound comprising:

-   i) a first fluorochrome having first absorption and emission    spectra; and-   ii) at least one of a second fluorochrome each said second    fluorochrome being covalently attached through a linker group to    said first fluorochrome and each second fluorochrome having second    absorption and emission spectra, the wavelength of the emission    maximum of the second fluorochrome(s) being longer that the emission    maximum of the first fluorochrome and a portion of the absorption    spectrum of each of said second fluorochromes overlapping a portion    of the emission spectrum of said first fluorochrome such that each    of said second fluorochromes is capable of accepting energy from    said first fluorochrome; and wherein said first fluorochrome    comprises a radical of the dye 4′,5′-bis-aminomethylfluorescein    having the structure of formula (II):

According to the first aspect, the fluorescein chromophore is employedas the donor fluorochrome in a fluorescent energy transfer labelingreagent. To this structure is covalently attached one or more secondfluorochromes. The one or more second fluorochromes are in an energytransfer arrangement with the first (or donor) fluorochrome, such thatphotoexcitation of a first fluorochrome results in the transfer ofenergy from that dye to the second acceptor fluorochrome(s).Furthermore, additional energy transfers involving one or moreadditional fluorochrome moieties may also be created. Thus, optionallyone or more third fluorochromes may be covalently attached to the“bifluorophore” complex by means of further linker groups. In thisarrangement, a portion of the emission spectrum of the secondfluorochrome overlaps the absorption spectra of the one or more thirdfluorochromes. The wavelength of the emission maximum of the thirdfluorochrome is longer that the wavelength of the second fluorochrome,such that energy absorbed by the first fluorophore(s) upon excitationwith light is transferred through the second to the third fluorophore togive an emission wavelength with a very large Stokes' shift.

Preferably, the reagent according to the first aspect includes at leastone target bonding group capable of forming a covalent bond with atarget material.

The linker group comprises a chain of linked atoms, suitably C₁₋₄₀ alkylchains, which may optionally include one or more groups selected from—C(O)—, —C(S)—, —NR′—, —O—, —S—, —CR′═CR′— and —CO—NR′— groups, where R′is hydrogen or C₁₋₄ alkyl. The chain may be optionally substituted, ifdesired, with groups as known to those skilled in the art which do notprevent energy transfer, for example, C₁₋₄ alkyl, C₁₋₄ alkoxy and halo.The linker group may include part of the constituents extending from thefluorochrome, that is, the linker groups may be derived from functionalgroups attached to the dye chromophore, suitably the 4′- and/or5′-aminomethyl groups and/or the 5(6)-carboxylic acid groups attached tothe fluorescein chromophore. Thus, while the linker is covalentlyattached to the dye chromophore, it is not a part of it. Furthermore,none of the linker groups should permit conjugation between donor andacceptor chromophores.

Fluorescent energy transfer labelling complexes according to the presentinvention show energy transfer ranging from 50% to 99% efficiency.Energy transfer efficiency depends on several factors such as spectraloverlap, spatial separation between donor and acceptor, relativeorientation of donor and acceptor molecules, quantum yield of the donorand excited state lifetime of the donor. In a preferred embodiment, thefluorochromes may be separated by a distance that provides efficientenergy transfer, preferably better than 75%.

In the present invention, the term “radical” is used to define the corestructure of the first fluorochrome and is derived from the dye,4′,5′-bis-aminomethylfluorescein (or its 5(6)-carboxylic acidderivative). Thus, 4′,5′-bis-aminomethylfluorescein forms the molecularbuilding block from which the fluorescent energy transfer reagents aresynthesised. Preferred positions for the covalent attachment of furtherfluorochromes, and optionally other substituents as defined herein areshown in FIG. 1. Furthermore, one or more hydrogen atoms of the aromaticring structures of the fluorescent dye-radical of formula (II) may bereplaced by a substituent group if desired, where the substituent isselected from a halogen (such as fluorine and chlorine), nitrile,hydroxy, thiol, C₁-C₆ alkyl, C₁-C₆ alkoxy and aryl.

The fluorescent energy transfer labelling reagents of the presentinvention preferably include a target bonding group capable of forming acovalent bond with a target material to enable the reagent to label thematerial, such as a biological compound. The target bonding group may belinked to the chromophore structure via a linker group, preferably (butnot exclusively) derived by chemical modification of the 4′- and/or5′-aminomethyl groups of 4′,5′-bis-aminomethyl-fluorescein. If4′,5′-bis-aminomethyl-fluorescein-5(6)-carboxylic acid is used as thedye building block, the 5- or 6-carboxylic moiety may also be chemicallymodified by well known methods so as to introduce a target bondinggroup. The target bonding group may be any group suitable for attachingthe dye to a target material, such as a carrier material, a biologicalcompound, or a further dye molecule. For example, the target bondinggroup may be a reactive group that can react under suitable conditionswith a complementary functional group of a target material.Alternatively, the target bonding group F may be a functional group andthe target may contain the reactive constituent. In either case, thetarget molecule becomes covalently labelled with the reagent accordingto the invention. Suitable reactive groups are selected fromN-hydroxysuccinimidyl ester, N-hydroxy-sulphosuccinimidyl ester,isothiocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonylhalide, acyl halide, anhydride and phosphoramidite. Suitable functionalgroups are selected from hydroxy, amino, sulphydryl, and carboxylgroups.

Suitably, the fluorescent energy transfer labelling reagent according tothe invention is a compound having the structure (III):

wherein:

-   D¹ is an acceptor dye selected from the group consisting of xanthine    dyes, rhodamine dyes and cyanine dyes;-   R¹ is selected from H, an amino-protecting group, the group -L²—F    and the group -L²-D², where D² is a fluorescent dye selected from    the group consisting of xanthine dyes, rhodamine dyes and cyanine    dyes;-   R², R³, R⁴ and R⁵ independently represent H, F, Cl, C₁-C₆ alkyl,    C₁-C₈ substituted alkyl, C₁-C₆ alkoxy, sulfonate, sulfone, amido,    nitrile, aryl or heteroaryl; or R² and R³ and/or R⁴ and R⁵ taken    together may be linked to form a fused aromatic or heteroaromatic    ring system;-   X¹, X², X³ and X⁴ independently represent H, F, Cl, C₁-C₆ alkyl,    C₁-C₆alkenyl, C₁-C₆ alkynyl, COOR′, SO₃H, CH₂OH, the group -L³—F and    the group -L³-D³, where D³ is a fluorescent dye selected from the    group consisting of xanthine dyes, rhodamine dyes and cyanine dyes;    and R′ is selected from hydrogen and C₁-C₄ alkyl;-   F is a target bonding group; and-   L¹, L² and L³ are each a linking group and each independently    comprises a group containing from 1 to 40 linked atoms selected from    carbon atoms which may optionally include one or more groups    selected from —C(O)—, —C(S)—, —NR′—, —O—, —S—, —CR′═CR′— and    —CO—NR′— groups, where R′ is hereinbefore defined.

Preferably, the compound of formula (II) includes at least one targetbonding group capable of forming a covalent bond with a target material.

Preferably, in the compound of formula (III), each of L¹, L² and L³independently contains from 1 to 20 atoms.

Preferably, L¹, L² and L³ are each independently:—{(CHR′)_(p)-Q-(CHR′)_(r)}_(s)—where Q is selected from: —CHR′—, —C(O)—, —C(S)—, —NR′—, —O—, —CR′═CR′—and —CO—NR′—; R′ is hydrogen or C₁-C₄ alkyl, each p is independently0-5, each r is independently 0-5 and s is 1 or 2.

Preferably, Q is selected from —CHR′—, —C(O) and —CO—NH—, where R′, p, rand s are hereinbefore defined.

Specific examples of reactive groups and their complementary functionalgroups are shown in Table 1. TABLE 1 Possible Reactive Groups andFunctional Groups Reactive Therewith Reactive Groups Functional GroupsSuccinimidyl esters primary amino, secondary amino Anhydrides, acidhalides primary amino, secondary amino, hydroxyl Isothiocyanate aminogroups Vinylsulphone amino groups Dichiorotriazines amino groupsHaloacetamides, maleimides thiols, imidazoles, hydroxyl, amines Carboxylamino, hydroxyl, thiols Phosphoramidites hydroxyl groups

Particularly suitable reactive groups which are useful for labelingtarget materials with available amino and hydroxyl functional groupsinclude:

Particularly suitable reactive groups which are useful for labelingtarget materials with available thiol functional groups include:

Suitable amino-protecting groups will be well known to the skilledperson and include N-alkyl and N-alkenyl derivatives such as N-methyl,N-^(t)butyl and N-allyl; carbamates, such as benzyl carbamate; andN-acyl derivatives, such as N-formyl, N-acetyl and N-benzoyl.Derivatives of the compounds of formula (III) that include amino-groupprotecting groups will be useful in the synthesis of energy transfer dyelabelling reagents based on the molecular building block,4′,5′-bis-aminomethyl-fluorescein, during attachment of the otherfluorophore(s), target bonding groups, solubilizing and charge carryingsubstituents.

Aryl is an aromatic substituent containing one or two fused aromaticrings containing 6 to 10 carbon atoms, for example phenyl or naphthyl,the aryl being optionally and independently substituted by one or moresubstituents, for example halogen, straight or branched chain alkylgroups containing 1 to 10 carbon atoms, aralkyl and C₁-C₆ alkoxy, forexample, methoxy, ethoxy, propoxy and n-butoxy.

Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring systemcontaining at least one and no more than 3 heteroatoms which may beselected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, straightor branched chain alkyl groups containing 1 to 10 carbon atoms, aralkyland C₁-C₆ alkoxy, for example, methoxy, ethoxy, propoxy and n-butoxy.

Halogen and halo groups are selected from fluorine, chlorine, bromineand iodine.

Preferred examples of xanthine dyes are selected from fluorescein,naphthofluorescein, rhodol and derivatives thereof.

Preferred examples of rhodamine dyes are selected from5-carboxyrhodamine (Rhodamine 110-5), 6-carboxyrhodamine (Rhodamine110-6), 5-carboxyrhodamine-6G (R6G-5 or REG-5), 6-carboxyrhodamine-6G(R6G-6 or REG-6), N,N,N′,N′-tetramethyl-5-carboxyrhodamine,N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or TMR),5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine (ROX).

Preferred examples of cyanine dyes are selected from Cy3(3-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-carbocyanine),Cy3.5 (3-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3disulphonato)dibenzo-carbocyanine), Cy5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-dicarbocyanine,Cy5.5(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)-dibenzo-dicarbocyanine,Cy7(1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-tricarbocyanine.

In one embodiment of the invention, the fluorescent labelling reagentsfurther comprise one or more water solubilizing substituents attachedcovalently to the dye chromophore, either directly or indirectly via asuitable linker group. The water solubilising constituents must beunreactive with the target bonding group of the complex. Solubilisinggroups, for example, sulphonate, sulphonic acid and quaternary ammonium,may be attached directly to aromatic ring structures of the dyechromophores. Alternatively, solubilising groups may be attached bymeans of a C₁ to C₆ alkyl linker chain to the aromatic ring structuresand may be selected from the group —(CH₂)_(k)-W where W is selected fromsulphonate, sulphonic acid, quaternary ammonium and carboxyl; and k isan integer from 1 to 6. Water solubility may be advantageous whenlabelling biological target materials, for example, proteins and nucleicacid derivatives.

Alternatively, a less hydrophilic polar form of the energy transferreagent may bind non-covalently to DNA by intercalation between the basepairs or by interaction in the minor groove of DNA. Such compounds maybe useful for DNA quantitation or localisation. In this embodiment, thefluorescent labelling reagents of the invention further comprise acharge carrying group, suitably a chain containing from 1 to 5positively charged nitrogen or phosphorus atoms. Some of thesepositively charged nitrogen or phosphorus atoms may be present in thelinker groups, L¹, L² and/or L³. Preferably, the charge carrying groupcontains positively charged nitrogen atoms, each provided by aquaternary ammonium group, or alternatively a protonated tertiary aminogroup, a guanidinium group, or a pyridinium group. A particularlypreferred charge carrying group is a straight or branched chaincontaining from 1 to 30 chain carbon atoms said group having thestructure:—(CH₂)_(m)N⁺R^(a)R^(a)R^(b)wherein each R^(a) is independently C₁-C₄ alkyl and R^(b) is C₁-C₄ alkylor is the group —(CH₂)_(m)N⁺R^(a)R^(a)R^(b) where R^(a) and R^(b) arehereinbefore defined and m is an integer from 1 to 4. The additionalcharge on the labeling complex allows the manipulation ofelectrophoretic mobility of target molecules labelled with the energytransfer reagents of the invention.

In another embodiment, the fluorescent labelling reagents furthercomprise two or more first fluorochromes linked in an energy transferrelationship with a second fluorochrome. As shown in FIG. 2, each of thefirst fluorochromes comprises a radical of the dye4′,5′-bis-aminomethylfluorescein-5(6)-carboxylic acid and the firstfluorochromes are covalently linked head to tail through the 4′- (or5′-)amino and the carboxyl groups of the radical. The first fluorochrome“complex” contains additional sites that may be utilised for covalentattachment of the second fluorochrome and/or a target material. Uponexcitation with light, the fluorescein donor molecules transfer thecombined energy absorbed to the second fluorochrome.

In addition to the embodiment of the invention which includes a singledonor and a single acceptor fluorochrome, the fluorescent energytransfer labelling reagents according to the invention may furthercomprise one or more third fluorochromes each having third absorptionand emission spectra covalently attached to said first or secondfluorochromes. For example, a third fluorochrome may be attached to asecond fluorochrome. In this example, the wavelength of the emissionmaximum of the third fluorochrome is longer than the wavelength of theemission maximum of the second fluorochrome. A portion of the absorptionspectrum of the third fluorochrome overlaps a portion of the emissionspectrum of the second fluorochrome such that excitation of said firstfluorochrome produces fluorescence from the third fluorochrome.

In a still further embodiment of the invention, the fluorescentlabelling reagent may contain a plurality of said second fluorochromes,each covalently attached through a linker to said first fluorochrome,each of said second fluorochromes being capable of accepting energy fromsaid first fluorochrome when said first fluorochrome is excited bylight. The extinction coefficient of the first fluorochrome is suitablygreater than 50,000 Liter/mole cm and the quantum yield greater than0.5, preferably greater than 0.75. Preferably, the second fluorophorehas an extinction coefficient of greater than 40,000 Liters/mole cm anda quantum yield of 0.1 or greater (compared to fluorescein as unity).Furthermore, the third fluorochrome(s), if employed in the labellingcomplex, should also have an extinction coefficient, preferably greaterthan 40,000 Liters/mole cm, as well as quantum yield of 0.1 or greater.In one embodiment, energy transfer from donor to acceptor chromophoresmay be achieved by exciting the fluorophore at 488 nm and then allowingthe energy transfer process to generate emission from the longestemitting fluorophore. Alternatively, in a cascade process, both donorand intermediate fluorophores may be excited simultaneously at 488 nm.The energy absorbed by both fluorophores is then transferred to a thirdfluorophore

In a still further embodiment, the energy transfer reagent may include achain of fluorescein polymers with acceptor fluorochromes, or otherfunctional groups, attached to different positions on the chain so as tosatisfy the requirements for different specific applications as shown inFIG. 3. The acceptors may be the same or may be different as required.

The labeling reagents of the invention are synthesized preferably bycovalently linking 4′,5′-bis-aminomethyl-fluorescein-5(6)-carboxylicacid to other fluorophores by known methods to form energy transferdonor-acceptor labelling reagents. The energy transfer reagents may beused to covalently label and thereby impart fluorescent properties totarget materials. Thus, in a second aspect, there is provided a methodfor labelling a target material, the method comprising adding to aliquid containing said target material a fluorescent energy transferreagent according to the present invention, and incubating said reagentwith the target material under conditions suitable for binding to andthereby labelling said target material. The method comprises incubatingthe target material with an amount of the energy transfer labellingreagent having at least one target bonding group as definedhereinbefore, under conditions to form a covalent linkage between thetarget and the labelling reagent. Suitable target biological materialsinclude, but are not limited to the group consisting of: antibodies,lipids, proteins, peptides, carbohydrates, nucleotides containing or arederivatized to contain one or more amino, sulphydryl, carbonyl,hydroxyl, carboxyl, phosphate or thiophosphate groups; oxy or deoxypolynucleic acids containing or are derivatized to contain one or moreamino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate orthiophosphate groups; microbial materials, drugs, hormones, cells, cellmembranes and toxins.

In an alternative embodiment, the fluorescent reagents need not have atarget bonding group and may be used to bind non-covalently to anothercompound. For example, the complex may be dissolved, then mixed in anorganic solvent with a polymer particle, such as polystyrene thenstirred by emulsion polymerization. The solvent is evaporated and thefluorescent dye complex is absorbed into the polystyrene particles.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood by reference to thedrawings in which:

FIG. 1 shows the structure of 4′, 5′ bis-aminomethyl-fluoresceinmolecular building block and the preferred positions for possibleattachment of the other fluorophore(s), target bonding groups,solubilizing and charge carrying substituents, and/or target material.

FIG. 2 shows the molecular structure of a dimeric4′,5′-bis-aminomethyl-fluorescein-5-carboxylic acid and the preferredpositions for possible attachment of the other fluorophore(s) and/orother target material.

FIG. 3 shows the molecular structure of a polymer of4′,5′-bis-aminomethyl-fluorescein-5-carboxylic acid and the positionsfor possible attachment of the other fluorophores(s) and/or other targetmaterial.

FIG. 4 is a schematic illustration of the overlapping absorption ( - - -), and emission ( - - - ) spectra of fluorophores suitable for FRET.

FIG. 5 shows the absorption and emission (excitation at 488 nm) spectraof FAM-Cy5 “bifluor” in MeOH/Hunig base.

FIG. 6 shows the absorption and emission (excitation at 488 nm) spectraof FAM-TAMRA-Cy5 “trifluor” in MeOH/Hunig base.

FIG. 7 is a Photon Flow Diagram for Donor-Acceptor Pair (BB). The flowdiagram monitors the fate of 100 photons absorbed by the fluoresceindonor in the donor-acceptor pair (BB).

FIG. 8 is a Photon Flow Diagram for Trifluor (TA).-Acceptor Pair (BB).The flow diagram monitors the fate of 100 photons absorbed by thefluorescein donor in the donor-acceptor pair (BB).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides fluorescent labeling reagents with largeStokes' shifts. For purposes of the present invention, the Stokes' shiftof the labelling reagent is the difference in nanometers between theabsorption maximum of the shortest wavelength light absorber of thereagent and the emission maximum of the longest wavelength emitter. Theenergy transfer labelling reagents as hereinbefore described, maycontain two or more fluorophores linked together for transfer of energyfrom a shorter wavelength absorber to a longer wavelength emitterresulting in a large Stokes' shift.

As shown schematically in FIG. 4, the shortest absorbing fluorophore,the first donor fluorophore, absorbs energy upon excitation at anexcitation wavelength (solid line) within its absorbance spectrum andemits energy at a wavelength within its emission spectrum (broken line).When linked at an appropriate distance and orientation to a secondfluorophore, the first fluorophore transfers, or donates, the energyfrom its excited state to the second fluorophore at the wavelengthwithin the absorption spectrum (solid line) of the second fluorophore.The second fluorophore accepts the donated energy and emits it at awavelength within its emission spectrum (broken line), which as shown,is longer in wavelength than the longest wavelength of the emission ofthe first fluorophore. This process is repeated until the emission forthe final, longest wavelength fluorophore ends the chain of energytransfer.

The amount of energy transferred from one fluorophore to the next, doesnot only depend on the overlap of the emission spectrum of the donor andthe absorption spectrum of the acceptor, as illustrated by the shadedarea between the first and second fluorophore, shown in FIG. 4.Forester's theory regarding resonance energy transfer predicts that theamount of energy transferred should depend on a spectral overlap termhaving a fourth power dependency on wavelength of the overlap region.Hence, the energy transfer is more efficient between fluorophores havinglonger absorption and emission wavelengths.

The fluorescent labeling reagents according to the invention have lowmolecular weights and can be readily conjugated to antibodies, otherproteins, and DNA probes. Low molecular weight as used herein shall meanthat the combined molecular weight of the labelling reagent is between500 and 10,000 Daltons. Therefore, these labeled species will have muchgreater penetration into intracellular environments than is possiblewith the larger phycobiliprotein labels currently in use. The lowmolecular weight fluorescent labeling reagents of the invention shouldbe valuable not only for flow cytometry, but also for laser confocalmicroscope and for other detection systems requiring multicolor todetection with single wavelength excitation.

The fluorescent labeling reagents preferably include groups capable offorming covalent bonds with corresponding groups on target compounds.Preferably, reactive groups are on the labelling reagent and functionalgroups are on the target compound or molecule. However, those skilled inthe art will is recognize that the functional group may be placed on thelabelling reagent and the reactive group may be on the target.

The fluorescent energy transfer dyes according to the present inventionmay be used in applications that include detecting and distinguishingbetween various components in a mixture. Thus, the invention alsoprovides a set of two or more different fluorescent energy transferlabelling reagents according to formula (III), wherein each labellingreagent in the set absorbs light energy of substantially the samewavelength and emits (or fluoresces) at a wavelength that isdistinguishable from every other reagent in the set. A set of reagentsincluding at least two labelling reagents of the invention may be usedin a multiparameter method for detecting target biological compoundspresent in multiple samples. The method comprises: a) incubating eachseparate sample with a different label from the set of fluorescentlabels to provide fluorescently-labelled samples; b) mixing each of saidfluorescently-labelled samples to form a single mixture containing allsamples; and c) irradiating the single mixture with a single wavelengthexcitation source and detecting the fluorescence emissions correspondingto each of the different fluorescently-labelled samples.

Examples of some of the dyes that can be used in the fluorescentlabeling reagents of the invention are shown in Table 2. These examplesare provided for illustration purposes only and other similar type ofdyes may also be used. The following examples should not be construed aslimiting the appended claims and the scope of the invention. The currentinvention should encompass any and all variations that become evidentfrom the teachings provided herein. TABLE 2 Absorption max (nm) Emissionmax (nm) Fluorophore (in MeOH) (in MeOH) Fluorescein 490 520 R110 503528 R6G 520 546 TAMRA 540 565 Cy3 ™ 550 570 ROX 568 595 Cy3.5 581 596TEXAS RED 583 603 Cy4 610 628 Cy5 649 670 Cy5.5 675 694 Cy7 743 767

EXAMPLES

1. Synthesis of 4′,5′-bis-Aminomethyl-fluorescein

1.1 4′,5′-bis-(2-Chloroacetamido)-aminomethyl-fluorescein

Fluorescein (3.3 grams) and 2-chloro-n-(hydroxymethyl)-acetamide (5.0grams) were dissolved in 20 ml of concentrated sulfuric acid. The darkbrown solution was stirred at room temperature for two hours. At suchtime, ESMS⁺ indicated that there was no starting fluorescein left. Theproduct was poured into 200 grams of ice and water and the precipitatewas filtered, washed with water, followed by ether and air-dried. NMR ofthe material, thus obtained, indicated that it was the desired product.

1.2 Hydrolysis of 4′,5′-bis-(2-Chloroacetamido)-aminomethyl-fluorescein

The product from the above reaction was suspended in 40 ml ofconcentrated hydrochloric acid and heated to reflux for 30 minutes. Aclear solution was obtained. The product was evaporated to dryness andthe residue recrystalized from methanol/dichloromethane to give thedesired product, 4′,5′-bis-aminomethyl-fluorescein, as identified by itsNMR and ESMS⁺

2. Synthesis of 4′,5′-bis-Aminomethyl fluorescein-5-carboxylic acid

Since fluorescein-5-carboxylic acid is only sparingly soluble inconcentrated sulfuric acid, a modified procedure was employed, asfollows.

2.1 4′,5′-bis-(2-Chloroacetamido)-aminomethyl fluorescein-5-carboxylicacid

To 20 ml of concentrated sulfuric acid, stirred at room temperatures,was added dipivaloyl-fluorescein-5-carboxylic acid. To the suspensionwas added, in portions, excess (4 equivalents) of2-chloro-n-(hydroxymethyl)-acetamide, until a clear solution wasobtained. More of the starting material (both fluorescein and excess2-chloro-n-(hydroxymethyl)-acetamide) was added until the color of thesolution turned from light yellow to brown. The solution was poured ontoan ice/water mixture. The precipitate thus obtained, was filtered,washed with water and ether. NMR and ESMS⁺ of the precipitate indicatedthat it was the desired product.

2.2 Hydrolysis of 4′,5′-bis-(2-Chloroacetamido)-aminomethylfluorescein-5-carboxylic acid

The product from 2.1 above (1.01 grams) was suspended in 20 ml ofconcentrated hydrochloric acid and 5 ml of 2-methoxyether. The resultingsuspension was heated to reflux and it began to clear in ca. two hours.The solution was, then, allowed to cool. After standing at roomtemperature overnight, massive precipitation occurred. The precipitatewas filtered, washed with 0.1 N hydrochloric acid followed by ether togive the desired product, 4′,5′ bis-aminomethyl fluorescein-5-carboxylicacid, as shown by its NMR and ESMS⁺ spectra.

3. Synthesis of Aminomethyl-FAM-Cy5 bifluor (BB)

4′-Aminomethyl-fluorescein (5 mg) was dissolved in 0.5 ml of dry DMF. Tothe solution was added 20 mg of Cy5 mono-functional reactive dye in 1.0ml of sodium bicarbonate-carbonate buffer. After 20 minutes at roomtemperature, the solvent was evaporated and the residue chromatographedto give the desired product (BB).

It will be readily appreciated that 4′,5′-bis-aminomethyl fluorescein(prepared by an analogous method as in Example 2) may be used in placeof 4′-aminomethyl-fluorescein to prepare 4′,5′-bis-aminomethyl-FAM-Cy5bifluor, which in turn may be used to prepare an energy transferlabelling reagent having a target bonding group attached at the free5′-aminomethyl position in the molecule. For example, as shown inreaction Scheme 1, treatment of 4′,5′-bis-aminomethyl-FAM-Cy5 bifluorwith succinic anhydride or glutaric anhydride in pyridine affords acarboxylic acid derivatised bifluor dye, which in turn may be convertedto its reactive N-hydroxysuccinimidyl ester derivative by reaction withN-hydroxysuccinimide/Dicyclohexyl-carbodiimide in DMF.

4. Synthesis of Aminomethyl FAM-TAMRA-Cy5 (TA)-“a trifluor”

The synthesis of trifluor, FAM-TAMRA-Cy5 involves the following steps.4.1. Aminomethyl FAM-TAMRA “bifluor”(BA)

4′,5′-bis-Aminomethyl-fluorescein (4 mg) and5-carboxytetra-methylrhodamine succinimidyl ester (10 mg) were dissolvedin 1 ml of dry dimethylformamide (DMF) with excessN,N-diisopropylethylamine. The reaction was allowed to proceed at roomtemperature overnight. The product was purified, by TLC, to give thedesired the bifluor (BA).4.2 Aminomethyl-FAM-TAMRA-Cy5 “trifluor” (TA)

The bifluor (BA), obtained in 4.1 above, was dissolved in drydimethylformamide with excess N,N-diisopropylethylamine added. To thesolution was added a slight excess of Cy-5 mono-functional NHS ester incarbonate/bicarbonate buffer. At the end of the reaction, as shown bythe disappearance of the starting material (BA) on thin layerchromatography (TLC), the solvent was evaporated to dryness and theresidue chromatographed on a C₁₈ reversed phase TLC plate to give thedesired product (TA).

Attachment of Trifluor (TA) to Biological Molecule

4′,5′-bis-aminomethyl fluorescein-5(6)carboxylic acid (prepared as inExample 2) may be used in place of 4′,5′-bis-aminomethyl-fluorescein toprepare 4′,5′-bis-aminomethyl-FAM-5(6)carboxylic acid-Cy5 “bifluor” orAminomethyl FAM-5(6) acid-TAMRA-Cy5 “trifluor”, which in turn may beused to prepare an energy transfer labelling reagent having a targetbonding group attached at the free 5(6)-position of donor fluorescein inthe molecule. For example, 4′,5′-bis-aminomethyl-FAM-5(6)carboxylicacid-TAMRA-Cy5 “trifluor” may be converted to its reactiveN-hydroxysuccinimidyl ester derivative by reaction withN-hydroxysuccinimide/Dicyclohexyl-carbodiimide in DMF which in turn maybe reacted with a target biological molecule.

5. Energy Transfer Measurements

5.1 Because of the complexity involved in the practical determination ofthe extent of energy transfer from a donor fluorochrome to an acceptorfluorochrome, even in a two-fluorophore case, no rigorous, reliablemethods for determining energy transfer have been published. In general,according to recent literature, “The efficiency of energy transfer wasestimated by calculating the amount of quenching of donor fluorescencethat occurs (DQE) when an acceptor is attached”. In another instance, acomparison of the “fluorescence strength” of the donor-acceptor pair wasobtained by comparing the intensity of the emission from the acceptor atits emission wavelength, upon excitation at the donor absorptionwavelength. Correction for the difference in donor concentration wasmade by measurement of the concentration of the donor-acceptor pair withabsorption at the donor absorption wavelength. This method offers ameans to compare the efficiency of energy transfer of donor-acceptorpairs; however, it is limited to the cases where the same donor isinvolved.

During investigations, it has been found that the first method ofestimation, based on DQE, routinely overstates the amount of energytransfer between donor and acceptor, since the amount of loss of energyby the donor is seldom completely transferred to the acceptor. Thesecond direct comparison method does not offer the flexibility of beingable to compare donor-acceptor pairs with different donors.

A new method has therefore been developed for determining the portion ofthe energy absorbed by the donor (and not emitted as donor emission)i.e. that which is transferred to the acceptor. This is the percentageof DQE which is actually emitted by the acceptor. The method involvesthe measurement of:

i) The absorption and emission characteristics of the donor and theacceptor in the non-conjugated states.

ii) The absorption spectra of the donor-acceptor pair. The opticaldensities (o.d.) are recorded at the donor absorption wavelength and theacceptor wavelength. For fluorescein-based donor-acceptor pairs, theo.d. at 488 nm is measured, since this wavelength is used to excite thedonor-acceptor pairs,

iii) The number of photons emitted by the donor measured at the donoremission wavelength and the number of photons emitted by the acceptormeasured at the acceptor emission wavelength upon excitation at thedonor absorption wavelength. For fluorescein-based donor-acceptor pairs,the excitation wavelength is 488 nm.

iv) The number of photons emitted by the acceptor in the donor-acceptorpair upon excitation at the acceptor absorption wavelength.

Definitions

i) A slope (SL): is the number of photons emitted, divided by the o.d.of the fluorophore being excited at the excitation wavelength. Sinceo.d. is directly proportional to number of photons absorbed, the slopeis proportional to the quantum yield, which is defined as the number ofphotons emitted divided by the photons absorbed. Typically, this numberis instrument-dependent, depending on its configuration, correctionsbeing required for the excitation source and photomultiplier efficiency.For a particular instrument with preset parameters, the slope measuredfor a reference compound, such as fluorescein, is close to being aconstant within experimental error.

ii) SLDFD is the slope of donor in its free state when excited at thedonor absorption wave-length,

iii) SLDCD is the slope of the donor in the donor-acceptor pair whenexcited is at the donor absorption wavelength,

iv) SLAFA is the slope of the acceptor in its free state, excited at theacceptor absorption wavelength,

v) SLACD is the slope of the acceptor in the donor-acceptor pair whenexcited at the donor absorption wavelength,

vi) SLACA is the slope of the acceptor in the donor-acceptor pair whenexcited at the acceptor absorption wavelength,

vii) PQEQ is the percentage of donor quenching, (=DQE¹¹),

viii) PEEA is the percentage quantum yield for the acceptor in thedonor-acceptor pair, as compared to that of the free acceptor whenexcited at the acceptor absorption wavelength, and

ix) PET is the percentage energy transfer of the energy absorbed by thedonor to be emitted by the acceptor in the donor acceptor pair whenexcited at the donor absorption wavelength.

The following calculations may be made.

-   1. PQEQ (1-SLDCD/SLDFD)×100%,-   2. PEEA=(SLACA/SLAFA)×100%-   3. PET=(Quantum yield of the donor)×SLACD/SLDFD.    5.2 Energy Transfer in the Bifluor, Aminomethyl FAM-Cy-5 (BB

As an example, the following values were obtained in the measurement ofenergy transfer in the donor-acceptor pair, aminomethyl FAM-Cy-5 (BB),(MeOH solvent+one drop of N,N-diisopropylethylamine).

-   SLDFD=1.41×10⁸-   SLAFA=1.39×10⁸-   SLDCD=1.12×10⁷-   SLACD=1.38×10⁷-   SLACA=1.46×10⁸

Thus:

-   -   PQEQ=92%    -   PEEA=106% and    -   PET=10% (with the quantum yield of fluorescein taken as 1.0).

A flow diagram, shown as FIG. 7, may be constructed from the abovevalues, which monitors the fate of 100 photons absorbed by thefluorescein donor in the donor-acceptor pair (BB) and showing thesignificance of the values. As can be seen from the flow diagram, of the92 photons loss to the donor upon the absorption of 100 photons, only 10are transferred to the acceptor. Thus, DQE, as described in U.S. Pat.No. 6,130,094 loc.cit, cannot be used to approximate the energy(photons) transferred to the acceptor.

5.3 Energy Transfer in the Trifluor. Aminomethyl FAM-Cv-5 (BB

The present method for determining energy transfer can be extended tomore than two fluorophores in an energy transfer fluorescent labellingreagent. As an example, the following results were obtained for theenergy transfer of the trifluor (TA) in MeOH with a trace ofN,N-diisopropylethylamine as solvent.

-   1) PQEQ of the first donor,-   2) PEEA of the first acceptor (PEEA₁),-   3) PET of the first donor to first acceptor (PET₁),-   4) PEEA of the second acceptor (PEEA₂),-   5) PET of the first donor to second acceptor (PET₂).    Thus:-   PEQE=90%,-   PEEA₁=0%, (No emission from the TAMRA fluorophore in (TA) was    observed by excitation either at the fluorescein absorption    wavelength or TAMRA absorption wavelength),-   PET₁=0%,-   PEEA₂=43%,-   PET₂=39%

An energy (photon) flow diagram may be constructed, as shown in FIG. 8.By comparing the energy transfer of (BB) and (TA), it can be seen thatthe introduction of the intermediate fluorophore, TAMRA, improves theenergy transfer from fluorescein to Cy5 by a factor of 4. Theimprovement was obtained as a result of better spectral overlaps insuccessive energy transfer steps over a single, direct one step energytransfer. Furthermore:

1) The quantum yield of the acceptor/the quantum yield of the donor isequal to the ratio SLAFA/SLDFD. Thus, if the donor is fluorescein, theratio SLAFA/SLDFD gives the quantum yield of the acceptor (relative tofluorescein as 1.0).

2) PETs as measured are actually the quantum yield of the donor-acceptorpairs excited at the donor absorption wavelength with the emissionmeasured as the acceptor emission maximum. This correlation can beapplied to a donor-acceptor pair with multiple acceptors.6. Synthesis of a Positively Charged Aminomethyl FAM-TAMRA(Donor-Acceptor Pair (BC)

The bifluor (BA) obtained before was dissolved in DMF and reacted with acarbonate/bicarbonate solution of the succinimidyl ester of the acid(CH₃)N⁺(CH2)₃N⁺(CH₃)2(CH₂)₃N⁺(CH₃)₂CH₂COOH at room temperature for 20minutes. The solvent was removed under vacuum and the residuechromatographed on C₁₈ reversed phase column to give the desiredproduct.

7. Synthesis of an Aminomethyl FAM-TAMRA-terminator (BD)

The preparation of 2′,3′-dideoxycytidine triphosphate labelled with anenergy transfer dye, involved the following steps (Reaction Scheme 2).

7.1 The 4′,5′-bis-aminomethyl fluorescein obtained as in Example 1 wasdissolved in DMF and reacted with succinic anhydride to give theprecursor (BE).

7.2 Compound (BE) was reacted with trifluoroacetic acid NHS ester in amixture of pyridine and dichloromethane to give the intermediate (BG).

7.3 Compound (BG) was dissolved in DMSO and reacted with appropriatelinker attached to dideoxycytidine triphosphate in acarbonate/bicarbonate buffer to give a crude product (BH).

7.4 The product obtained in 7.3 was purified and reacted with theTAMRA-NHS ester to give (BF), the final product, which was purified byreverse phase HPLC.

Those skilled in the art having the benefit of the teachings of thepresent invention as set forth above, can effect numerous modificationsthereto. These modifications are to be construed as being encompassedwithin the scope of the present invention as set forth in the appendedclaims.

1. A compound comprising: i) a first fluorochrome having first absorption and emission spectra; and ii) at least one of a second fluorochrome each said second fluorochrome being covalently attached through a linker group to said first fluorochrome and each second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of the second fluorochrome(s) being longer than the emission maximum of the first fluorochrome and a portion of the absorption spectrum of each of said second fluorochromes overlapping a portion of the emission spectrum of said first fluorochrome such that each of said second fluorochromes is capable of accepting energy from said first fluorochrome; and wherein said first fluorochrome comprises a radical of the dye 4′,5′-bis-aminomethylfluorescein having the formula:


2. The compound according to claim 1 wherein said compound includes at least one target bonding group capable of forming a covalent bond with a target material.
 3. The compound according to claim 1 or 2 further comprising: charge carrying substituents or water solubilizing substituents, covalently attached thereto, or charge carrying and water solubilizing substituents, covalently attached thereto, said substituents being unreactive with said target bonding group.
 4. The compound according to claim 3 wherein said water solubilizing substituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.
 5. The compound according to claim 3 wherein said charge carrying substituents incorporate from one to five positively charged nitrogen or phosphorus atoms.
 6. The compound according to claim 2 wherein said target bonding group is a reactive group selected from the group consisting of N-hydroxysuccinimidyl ester, N-hydroxy-sulphosuccinimidyl ester, isothiocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, acyl halide, anhydride and phosphoramidite.
 7. The compound according to claim 2 wherein said target bonding group is a functional group selected from the group consisting of amino, hydroxyl, sulphydryl, and carboxyl groups.
 8. The compound according to claim 1 wherein each of said second fluorochromes are selected from xanthine dyes, rhodamine dyes and cyanine dyes.
 9. The compound according to claim 2 wherein said target material is selected from the group consisting of: antibodies, lipids, proteins, peptides, carbohydrates nucleotides containing or are derivatized to contain one or more amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate, or thiophosphate groups; oxy or deoxy polynucleic acids containing or are derivatized to contain one or more amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate, or thiophosphate groups; microbial materials, drugs, hormones, cells, cell membranes and toxins.
 10. The compound according to claim 1 having a plurality of said second fluorochromes each covalently attached through a linker to said first fluorochrome, and each of said second fluorochromes being capable of accepting energy from said first fluorochrome when said first fluorochrome is excited by light.
 11. The compound according to claim 1 further comprising two or more first fluorochromes linked in an energy transfer relationship with a second fluorochrome and wherein each said first fluorochrome comprises a radical of the dye 4′,5′-bis-aminomethylfluorescein-5(6)-carboxylic acid and said two or more first fluorchromes being covalently linked head to tail through the 4′-(or 5′-)amino and carboxyl groups of said radical.
 12. The compound according to claim 1 further comprising one or more third fluorochromes covalently attached to said first or second fluorochromes, and each third fluorochrome having third absorption and emission spectra, the wavelength of the emission maximum of said third fluorochrome(s) being longer than the wavelength of the emission maximum of said second fluorochrome and a portion of the absorption spectrum of each of said third fluorochrome(s) overlapping a portion of the emission spectrum of said second fluorochrome such that excitation of said first fluorochrome produces fluorescence from said third fluorochrome(s).
 13. A compound having the structure:

wherein: D¹ is an acceptor dye selected from the group consisting of xanthine dyes, rhodamine dyes and cyanine dyes; R¹ is selected from H, an amino-protecting group, the group -L²—F and the group -L²-D², where D² is a dye selected from the group consisting of xanthine dyes, rhodamine dyes and cyanine dyes; R², R³, R⁴ and R⁵ independently represent H, F, Cl, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₁-C₆ alkoxy, sulfonate, sulfone, amido, nitrile, aryl or heteroaryl; or R² and R³ and/or R⁴ and R⁵ taken together may be linked to form a fused aromatic or heteroaromatc ring system; X¹, X², X³ and X⁴ independently represent H, F, Cl, C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, COOR′, SO₃H, CH₂OH, the group -L³—F and the group -L³-D³, where D³ is a dye selected from the group consisting of xanthine dyes, rhodamine dyes and cyanine dyes; and R′ is selected from hydrogen and C₁-C₄ alkyl; F is a target bonding group; and L¹, L² and L³ are each a linking group and each independently comprises a group containing from 1 to 40 linked atoms selected from carbon atoms which may optionally include one or more groups selected from —C(O), —C(S)—, —NR′—, —O—, —S—, —CR′═CR′— and —CO—NR′— groups, where R′is hereinbefore defined.
 14. The compound according to claim 13 wherein said compound includes at least one target bonding group capable of forming a covalent bond with a target material.
 15. The compound according to claim 13 or 14 further comprising: charge carrying or water solubilizing substituents covalently attached thereto, or charge carrying and water solubilizing substituents covalently attached thereto, said substituents being unreactive with said target bonding group.
 16. The compound according to claim 15 wherein said water solubilizing substituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.
 17. The compound according to claim 15 wherein said charge carrying substituents incorporate from two to five positively charged nitrogen atoms.
 18. The compound according to claim 13 wherein each of L¹, L² and L³ independently contains from 1 to 20 atoms.
 19. The compound according to claim 13 wherein L¹, L² and L³ are each independently: —{(CHR′)_(p)-Q-(CHR′)_(r)}s— where Q is selected from: —CHR′—, —C(O)—, —C(S)—, —NR′—, —O—, —CR′═CR′— and —CO—NR′—; R′ is hydrogen or C₁-C₄ alkyl, each p is independently 0-5, each r is independently 0-5 and s is 1 or
 2. 20. The compound according to claim 19 wherein Q is selected from —CHR′—, —C(O)— and —CO—NH—, where R′, p, r and s are hereinbefore defined.
 21. The compound according to claim 14 wherein said target bonding group comprises a reactive group for reacting with a functional group on a target material, or a functional group for reacting with a reactive group on a target material.
 22. The compound according to claim 21 wherein said reactive group is selected from the group consisting of N-hydroxysuccinimidyl ester, N-hydroxy-sulphosuccinimidyl ester, isothiocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, acyl halide, anhydride and phosphoramidite.
 23. The compound according to claim 21 wherein said functional group is selected from the group consisting of amino, hydroxyl, sulphydryl, and carboxyl groups.
 24. The compound according to claim 13 wherein said xanthine dye is selected from fluorescein, naphthofluorescein, rhodol and derivatives thereof.
 25. The compound according to claim 13 wherein said rhodamine dye is selected from 5-carboxyrhodamine (Rhodamine 110-5), 6-carboxyrhodamine (Rhodamine 110-6), 5-carboxyrhodamine-6G (R6G-5 or REG-5), 6-carboxyrhodamine-6G (R6G-6 or REG-6), N,N,N′,N′-tetramethyl-5-carboxyrhodamine, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA or TMR), 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine (ROX).
 26. The compound according to claim 13 wherein said cyanine dye is selected from Cy3 (3-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-carbocyanine), Cy3.5 (3-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′tetramethyl-4,5,4′,5′-(1,3-disulphonato)dibenzo-carbocyanine), Cy5 (1-(s-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-dicarbocyanine, Cy5.5 (1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-4,5,4′,5′-(1,3-disulphonato)-dibenzo-dicarbocyanine, Cy7 (1-(ε-carboxypentyl)-1′-ethyl-3,3,3′,3′-tetramethyl-5,5′-disulphonato-tricarbocyanine.
 27. The compound according to claim 14 wherein said target material is selected from the group consisting of: antibodies, lipids, proteins, peptides, carbohydrates, nucleotides containing or are derivatized to contain one or more amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate or thiophosphate groups; oxy or deoxy polynucleic acids containing or are derivatized to contain one or more of an amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate or thiophosphate groups; microbial materials, drugs, hormones, cells, cell membranes and toxins.
 28. A method for labelling a target material comprising: a) adding to a liquid containing said target material a fluorescent energy transfer reagent according to claim 1 or claim 13; and b) incubating said reagent with said target material under conditions suitable for binding to and thereby labelling said target material.
 29. The method according to claim 28 wherein said target material is selected from the group consisting of: antibodies, lipids, proteins, peptides, carbohydrates, nucleotides containing or are derivatized to contain one or more amino, sulphydryl, carbonyl, hydroxyl, carboxyl, phosphate or thiophosphate groups; oxy or deoxy polynucleic acids containing or are derivatized to contain one or more amino, sulphydryl, carbonyl, hydroxyl carboxyl, phosphate or thiophosphate groups; microbial materials, drugs, hormones, cells, cell membranes and toxins. 